Study Of The Pharmacokinetics And Bioequivalence Of Tacrolimus In Chinese Healthy Volunteers

OBJECTIVE:A sensitive validated high performance liquid chromatography tandem mass spectrometric method(LC-MS/MS) for tarclimus in human blood has been developed and applied to a bioequivalence(BE) study.METHODS:According to the method of literatures,a sensitive,specific and simple LC-MS/MS method had been improved.The blood sample was prepared by a liquid-liquid extraction method.In a 1.5 mL centrifuge tube an aliquot 100μL blood by haemolysis was spiked with 10μL IS(CsD) solution and 150μL sodium fluoride solution.After vortexing,1mL of extraction solvent(ether) was added to the tube and the tube were vortex mixed for 1min.After centrigation at 3000 rpm for 5min,the organic phase was the transferred into another tube and evaporated to dryness under a gentle stream of nitrogen.The dry residue was dissolved in 100μL 70%acetonitrile solution.After centrifugation,10μL of supernatant fluid were injected into the LC-MS/MS system for analysis.Tacrolimus and the internal standard Cyclosporin D were chromatographed on a Thermo Hypersil Silica column(2.1×150mm,5μm) with the mobile phase consisted of acetonitrile-water(98:2,v/v,containing 20mM ammonium acetate and 0.1%formic acid).The flow rate was set 0.3 mL/min.Detection was performed on a triple quadrupole tandem mass spectrometry by multiplc rcaction monitoring(MRM) mode via negative eletrospray ionization(ESI) source.The mass transition ion-pair has been followed as m/z 821.5→768.4 for FK506 and m/z 1233.8→1216.7 for CsD.A single oral dose(5mg of tested and reference preparations) were given to 22 healthy volunteers in a randomized,open-label,two-period crossover design study. Blood sampling was conducted consequently within 72 hours.The pharmacokinetic parameters(AUC_0-72,AUC_0-8,C_max and T_1/2) were calculated and the bioavailability and bioequivalence of two preparations were evaluated by DAS 2.0 program.RESULTS:The assay exhibited good linearity over a working range of 0.20～100.0 ng/mL for tacrolimus in human blood with a lower limit of quantitation of 0.20 ng/mL.No endogenous compounds were found to interfere with the analysis.The accuracy and precision were shown for concentrations over the standard ranges.This method was successfully applied for the PK and BE studies by analysis of blood samples after an oral dose of 5 mg of tacrolimus in 22 healthy volunteers.After a single dose,the pharmacokinetic parameters of tacrolimus were as follows: Cmax were(49.41±22.21) ng/mL and(41.30±19.63) ng/mL,Tmax were(1.20±0.40) h and(1.70±0.77) h,AUC_0-72 were(366.996±191.246) ng·h/mL and(330.274±143.473) ng·h/mL;AUC_0-8 were(437.017±239.350) ng·h/mL and(394.944±186.339) ng·h/mL for T and R respectively.The inter-formulation of tested and reference preparations was not significant different(p＞0.05).The 90%confidential intervals of Cmax,AUC_0-72 and AUC_0-8 were(97.1～142.3)%,(86.0～124.4)%and(84.5～123.4)%.The relative bioavailability was(114.754±48.690)%by AUC_0-72.CONCLUSION:The method for determination of tarclimus concentration in blood is of high sensitivity,good selectivity and reproducibility.The results of the statistic analysis showed that the two preparations were bioequivalent.